Erythroid cells producing adult-type β-hemoglobin generated from human embryonic stem cells

ABSTRACT

Methods and compositions of erythroid cells that produce adult β-hemoglobin, generated by culturing CD31+, CD31+/CD34+ or CD34+ cells from embryonic stem cells under serum-free culture conditions.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional PatentApplication No. 60/743,264, filed Feb. 9, 2006, incorporated herein byreference as if set forth in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under DOD ARPADAMD17-02-C-0130. The government has certain rights in this invention.

BACKGROUND

The present invention relates generally to methods and compositions oferythroid cells generated from human embryonic stem cells (hESCs). Moreparticularly, the present invention relates to methods and compositionsof erythroid cells that are at least CD31−, CD34−, CD71+ and CD235a+,and express adult β-hemoglobin and fetal γ-hemoglobin, but not embryonicζ-hemoglobin.

Hematopoiesis is a formation of blood cell components from stem cells,typically hematopoietic stem cells. Prenatally, hematopoiesis occurs inthe yolk sack, then the liver, and eventually the bone marrow. In normaladults, however, it occurs in bone marrow and lymphatic tissues. It hasbeen estimated that there is approximately 1 hematopoietic stem cell per10⁴ bone marrow cells.

The blood cells produced during hematopoiesis are divided into thefollowing three cell lineages: (1) erythroid cells, (2) lymphoid cells,and (3) myeloid cells. Erythroid cells, including normoblasts,erythroblasts and mature red blood cells (RBCs), are the most commontype of blood cell and are a principal means of delivering oxygen fromthe lungs to body tissues. Lymphoid cells, including B-cells andT-cells, are a type of white blood cell that play a significant role inthe body's immune defenses. Myeloid cells, including granulocytes,megakaryocytes, and macrophages, are a diverse group of cells comprisingother white blood cells (e.g., neutrophils, eosinophils and basophils)and platelets. Of particular interest herein is the generation of cellsof the erythroid lineage.

“Erythropoiesis” is a formation of erythroid cells from stem cells,typically from hematopoietic stem cells. In an average adult, productionof mature RBCs (erythrocytes) equals their loss. As such, the averageadult produces 3.7×10¹¹ RBCs/day.

Given the paucity of hematopoietic stem cells, researchers have recentlyshifted their attention to generating RBCs from embryonic stem cells(ESCs), especially hESCs. hESCs offer an opportunity to generate RBCs insufficient quantities to study the differentiation of RBCs in vitro.More importantly, RBCs generated from hESCs would provide a safe and anample alternative source of cells for transfusion, as well as fortreating conditions involving defective RBCs (e.g., hypoxia and sicklecell anemia). In the United States, for example, only five percent ofeligible donors across the nation donate blood; however, the number oftransfusions nationwide increases by nine percent every year.

Recently, Umeda et al. showed that primate ESCs co-cultured with OP9stromal cells generated cells that expressed embryonic, fetal and adulthemoglobin. Umeda K, et al., “Sequential analysis of alpha- andbeta-globin gene expression during erythropoietic differentiation fromprimate embryonic stem cells,” Stem Cells 24:2627-2636 (2006). However,Umeda et al. cultured the cells in serum, which may be problematic dueto the uncharacterized composition and variation of serum. Moreover,erythroid cells generated by Umeda et al.'s method contained 5% to 15%myeloid cells.

Likewise, Olivier et al. showed that hESCs co-cultured with human fetalliver cells generated CD34+ cells that produced embryonic and fetalhemoglobin. Olivier E, et al., “Large-scale production of embryonic redblood cells from human embryonic stem cells,” Exp. Hematol. 34:1635-1642(2006); for similar results, see also Qiu C., et al., “Differentiationof human embryonic stem cells into hematopoietic cells by coculture withhuman fetal liver cells recapitulates the globin switch that occursearly in development,” Exp. Hematol. 33:1450-1458 (2005). Unfortunately,Olivier et al.'s cells did not produce adult hemoglobin and retainedexpression of embryonic ζ-hemoglobin.

Other researchers have also generated RBCs from ESCs; however, thesemethods either used non-human/non-primate stem cells or used an embryoidbody-dependent method (i.e. no direct differentiation). These methods,however, produced a mixture of erythroid and myeloid cells. See CarottaS, et al., “Directed differentiation and mass cultivation of pureerythroid progenitors from mouse embryonic stem cells,” Blood104:1873-1880 (2004); Chadwick K, et al., “Cytokines and BMP-4 promotehematopoietic differentiation of human embryonic stem cells,” Blood102:906-915 (2003); Kaufman D, et al., “Hematopoietic colony-formingcells derived from human embryonic stem cells,” Proc. Natl. Acad. Sci.USA 98:10716-10721 (2001); Ng, E, et al., “Forced aggregation of definednumbers of human embryonic stem cells into embryoid bodies fostersrobust, reproducible hematopoietic differentiation,” Blood 106:1601-1603(2005); and Zambidis E, et al., “Hematopoietic differentiation of humanembryonic stem cells progresses through sequential hematoendothelial,primitive, and definitive stages resembling human yolk sac development,”Blood 106:860-870 (2005).

For the foregoing reasons, there is a continuing need for alternativemethods of generating erythroid cells from hESCs, especially erythroidcells that express adult hemoglobin, that are generated underplasma/serum-free conditions and that are free of lymphocytes.

BRIEF SUMMARY

In a first aspect, a method of culturing human embryonic stem cells intoerythroid cells producing adult-type β-hemoglobin includes isolatinghuman embryonic stem cell-derived cells that are CD31+, CD34+, andCD31+/CD34+ cells, such that the cells are enriched in CD31+/CD43+,CD31+/CD34+, CD34+/CD43+ or CD31+/CD34+/CD43+ hematopoietic progenitors.The method also includes culturing the hematopoietic progenitors tocause an expansion of erythroid precursor cells. The method furtherincludes recovering a population of erythroid cells, such thatessentially all of the live cells are erythroid cells that produce adultβ-hemoglobin, but not embryonic ζ-hemoglobin.

In some embodiments of the first aspect, the population of erythroidcells are essentially free of lymphocytes. In other embodiments of thefirst aspect, the erythroid cells contain less than 0.1% leukocytes.

In some embodiments of the first aspect, the hematopoietic progenitorsare cultured in the presence of stem cell factor and erythropoietinunder conditions preventing cell adherence.

In some embodiments of the first aspect, the human embryonic stem cellsare co-cultured with stromal cells to produce the hematopoieticprogenitors.

In some embodiments of the first aspect, at least 0.2×10⁵ erythroidcells are generated from one human embryonic stem cell. In otherembodiments of the first aspect, up to 2.0×10⁵ erythroid cells aregenerated from one human embryonic stem cell.

In some embodiments of the first aspect, the erythroid cells produceadult-type β-hemoglobin, are CD31−, CD34−, CD71+, CD235a+ andadditionally express fetal γ-hemoglobin. In other embodiments of thefirst aspect, the erythroid cells do not express embryonic ζ-hemoglobin.

In some embodiments of the first aspect, the purity of the isolatedhematopoietic progenitors are greater than 95% at a single column runand cell viability, as evaluated by Trypan blue exclusion, is higherthan 95%.

In some embodiments of the first aspect, the CD31+/CD34+ cells comprise30% to 50% CD31+/CD34+/CD43+ hematopoietic progenitors and 50% to 70%CD31+/CD34+/CD43−/KDR^(bright) endothelial cells.

In some embodiments of the first aspect, the cells express adultβ-hemoglobin by 10 days (±10%) of culture, and most of the cells have aphenotype and a morphology of erythroid cells.

In some embodiments of the first aspect, after approximately 30 days(±10%) of culture, essentially all of the live erythroid cells (>95%)show positive stains with antibodies against fetal γ-hemoglobin andadult β-hemoglobin, but no positive staining with antibodies againstembryonic ζ-hemoglobin. In other embodiments of the first aspect, afterapproximately 50 days (±10%) of culture, the erythroid cells areCD31−/CD34− and show adult β-hemoglobin expression and no embryonicζ-hemoglobin expression.

In some embodiments of the first aspect, the hematopoietic precursorsare cultured in serum-free culture conditions.

In a second aspect, a cell population includes a population of cells,such that essentially all of the live cells are erythroid cells thatproduce adult β-hemoglobin, but not embryonic ζ-hemoglobin.

In some embodiments of the second aspect, the population of essentiallyall live cells are nucleated erythroid cells. In other embodiments ofthe second aspect, the population is essentially free of lymphocytes. Infurther embodiments of the second aspect, the population contains lessthan 0.1% leukocytes. In alternative embodiments of the second aspect,the population is CD31−, CD34−, CD71+ and CD235a+.

In a third aspect, a preparation of cells includes CD31+/CD34+/CD43+hematopoietic progenitor cells.

In some embodiments of the third aspect, the preparation of cells are aresult of 6 to 7 days (±10%) co-culture between human embryonic stemcells and OP9 cells.

In a fourth aspect, a CD31+ cell population includes 10% to 20%CD31+/CD43+ hematopoietic progenitors, up to 60%CD31+/CD43−/KDR^(bright) endothelial cells and less than 15%CD34+/CD43−/KDR-mesenchymal cells.

In a fifth aspect, a CD34+ cell population includes 10% to 20%CD34+/CD43+ hematopoietic progenitors, up to 60%CD34+/CD43−/KDR^(bright) endothelial cells and less than 15%CD34+/CD43−/KDR-mesenchymal cells.

BRIEF DESCRIPTION OF DRAWINGS

The invention will be better understood and features, aspects andadvantages other than those set forth above will become apparent whenconsideration is given to the following detailed description thereof.Such detailed description makes reference to the following drawings,wherein:

FIG. 1 shows results from flow cytometric analysis of hESC-derivederythroid cells at differentiation day 5, 10 and 50 of culture;

FIG. 2 shows morphology of erythroid precursors generated fromhESC-derived CD34+ progenitors after two weeks of culture; and

FIG. 3 shows a diagram that summarizes one embodiment of the generationof hESC-derived erythroid cells.

While the invention is susceptible to various modifications andalternative forms, specific embodiments thereof have been shown by wayof example in the drawings and are herein described in detail. It shouldbe understood, however, that the description herein of specificembodiments is not intended to limit the invention to the particularforms disclosed, but on the contrary, the intention is to cover allmodifications, equivalents, and alternatives falling within the spiritand scope of the invention as defined by the appended claims.

BRIEF DESCRIPTION OF THE INVENTION

In one embodiment, the present invention is a generation of a cellpopulation of erythroid cells producing adult-type β-hemoglobin fromhESCs. By “erythroid cells producing adult-type β-hemoglobin,” we meancells that are CD31−. CD34−, CD71+, CD235a+ and that express adultβ-hemoglobin and fetal γ-hemoglobin, but not embryonic ζ-hemoglobin, asdetermined by PCR and flow cytometry using hemoglobin-specificantibodies. Morphologically, the cell population consists of nucleatederythroid cells at different stages of maturation, including, but notlimited to, normoblasts and erythroblasts. The population contains lessthen 0.1% leukocytes as determined by staining with anti-CD45 monoclonalantibodies and is essentially free of lymphocytes. By “essentially freeof lymphocytes,” we mean that the lymphoid cells cannot be detectedusing flow cytometry with monoclonal antibodies against B-cells (CD19),T-cells (CD3) and NK cells (CD94), or by RT-PCR through amplification ofB-cell (V-preB), T-cell (pre-Tα) and NK cell (CD94) specifictranscripts.

It is an advantage that the hESC-derived erythroid cells, if prepared bythe methods described herein, have not been exposed to blood plasma orserum. In a preferred embodiment, the cells of the present invention are“plasma free” and have never been exposed to human, or any other type,of blood plasma or serum.

It is also an advantage that the hESC-derived erythroid cells producedby the methods described herein can be used to produce younger cells(i.e. normoblasts and erythroblasts) that have prolonged survival. Thistrait would be beneficial for patients who require multipletransfusions, for example chronic anemia patients.

It is also an advantage that the hESC-derived erythroid cells producedby the methods described herein express adult hemoglobin followingco-culture (i.e. after selection of CD31+ and/or CD34+ hematopoieticprogenitor cells).

It is also an advantage that the hESC-derived erythroid cells can beproduced by the methods described herein free of viruses to reduce therisk of CMV, HTLV-I/II and prion (Creutzfeldt-Jakob disease, and newvariant Creutzfeldt-Jakob disease) transmission.

It is also an advantage that the hESC-derived erythroid cells are freeof lymphocytes. This is beneficial for use in immunosuppressedindividuals who are at increased risk of developing graft-versus-hostreaction after transfusion.

It is also an advantage that at least 0.2×10⁵ to preferably 2.0×10⁵erythroid cells are generated from one human embryonic stem cell.

We had previously isolated different types of hematopoietic precursorsfrom hESC/OP9 co-culture: CD31+, CD34+, CD235a+, CD43+ lin- and CD45+lin-cells. Vodyanik M, et al., “Human embryonic stem cell-derived CD34+cells: efficient production in the coculture with OP9 stromal cells andanalysis of lymphohematopoietic potential,” Blood 105:617-626 (2005);see also Vodyanik M, et al., “Leukosialin (CD43) defines hematopoieticprogenitors in human embryonic stem cell differentiation cultures. Blood108:2095-2105 (2006), each of which is incorporated herein by referenceas if set forth in its entirety. All of these hematopoietic precursorcells can be directly differentiated into RBCs; however, the bestexpansion of erythroid cells was achieved when we used hESC-derivedCD31+ or CD34+ hematopoietic progenitors as described below.

Several publications describe differentiation of ESCs into a mixture ofhematopoietic cells (Chadwick et al., supra; Ng et al., supra; andZambidis et al., supra), but there is no description of directeddifferentiation of hESCs to erythroid cells that are at least CD31−,CD34−, CD71+ and CD235a+ and that express adult β-hemoglobin. Other workhas described directed differentiation of mouse ESCs (Carotta et al.,supra) and human somatic CD34+ cells into RBCs. Giarratana M, et al.,“Ex vivo generation of fully mature human red blood cells fromhematopoietic stem cells,” Nat. Biotechnol. 23:69-74 (2005).

Recent work describes differentiation of hESCs into hematopoietic cellscontaining RBCs using a bone marrow stromal cell line (S17) and a humanfetal liver-derived cell line (FH-B-hTERT). However, RBCs obtained inthese systems produced embryonic ε-hemoglobin and fetal γ- andζ-hemoglobin, but failed to express adult β-hemoglobin (Qiu et al.,supra).

In one embodiment of the present invention, erythroid cells are obtainedby the following method (see FIG. 3):

One preferably first differentiates hESCs into CD31+ and/or CD34+ cells.We have used H1 and H9 hESCs (WiCell; Madison, Wis.) for the examplesdescribed below, but any hESC line is suitable. The best expansion wasachieved when we used CD34+ cells that comprised 10% to 20% ofCD34+/CD43+ hematopoietic progenitors, and up to 60% ofCD34+/CD43−/KDR^(bright) endothelial cells and less than 15% ofCD34+/CD43−/KDR-mesenchymal cells, or CD31+ cells that comprised 30% to50% CD31+/CD34+/CD43+ hematopoietic progenitors, and up to 50% to 70%CD31+/CD34+/CD43−/KDR^(bright) endothelial cells.

Preferably, one begins by co-culturing hESCs with a stromal cell line,such as mouse OP9 bone marrow stromal cells. In addition, the stromalcell line should not express macrophage colony stimulating factor. Otherbone marrow stromal lines are suitable as long as the line results inefficient production of CD31+, CD31+/CD34+ or CD34+ hematopoieticprogenitors.

By “efficient,” we mean that at least 5% to 10% CD31+ and/or CD34+ cellsare generated and that these cells comprise at least 30% to 50%CD31+/CD34+/CD43+ hematopoietic progenitors, and up to 70%CD31+/CD34+/KDR^(bright)/CD43− endothelial cells.

ESC differentiation into hematopoietic progenitors has been described byus and others. Vodyanik et al., supra; and Kaufman et al., supra. Onecould also obtain hematopoietic progenitors from embryoid bodies (see,e.g., Chadwick et al., supra; Zambidis et al., supra; and Ng et al.,supra.).

Preferably, the hESCs are added to OP9 cultures at a density of1.5×10⁶/20 ml (±10%) per 10 cm dish, in α-MEM (minimal essential media;GIBCO) supplemented with 10% fetal bovine serum (FBS; HyClone; Logan,Utah) and 100 μM monothioglycerol (MTG; Sigma; St. Louis, Mo.). Theexample below describes a preferable density of the hESCs.

The hESC/OP9 co-cultures are incubated for up to 10 days at 37° C. innormoxic conditions and 5% CO₂, with a half-medium change on day 4. Asingle-cell suspension is prepared on day 6 of culture. The cells areharvested and single cell suspensions are prepared by treatment of theco-culture with collagenase IV, typically at 1 mg/ml (±10%) in α-MEM for20 minutes at 37° C. followed by treatment with 0.05% trypsin-0.5 mMEDTA (Invitrogen) for 15 minutes at 37° C.

At this point, one would typically have a single cell suspension ofcells that contains at least 5% to 10% CD31+ cells, preferably asmeasured by anti-CD31 monoclonal antibodies. For example, in a typicalisolation of CD31+ cells using paramagnetic antibodies, the purity ofthe isolated CD31+ cells is greater than 95% at a single column run andcell viability, as evaluated by Trypan blue exclusion, is typicallyhigher than 95%. By “single column run,” we mean a single round of cellpurification using magnetic beads. Likewise, cells could be assayed withanti-CD34 monoclonal antibodies.

The conditions described herein provide the best hematopoieticdifferentiation (hematopoietic cell output) of which we are aware. Weknow from previous studies (Vodyanik et al., supra) that by day 7 we getthe highest number of erythroid progenitors in OP9 co-culture, includingdefinitive erythroid progenitors as determined using colony-formingassay followed by analysis of hemoglobin expression in colonies by PCR.

To expand and differentiate the erythroid cells, CD31+ and/or CD34+cells are typically cultured in serum-free expansion media (SFEM medium;Stem Cell Technologies; Vancouver, Canada) supplemented with,preferably, 0.3% (±10%) EX-CYTE® (Serologicals Proteins, Inc.; Kankakee,Ill.), 1 mg/ml (±20%) iron-saturated transferrin (Sigma), 10⁻⁶ M (±10%)dexamethasone, and 20 ng/ml (±10%) insulin in tissue culture flaskscoated with a substance to prevent cell adherence, such as poly2-hydroxyethyl methacrylate (HEMA; Sigma). For the first 5 days, cellsare typically cultured in the presence of 50 ng/ml (±10%) stem cellfactor (SCF), 2 U/ml (±10%) erythropoietin (EPO), 50 ng/ml (±10%)thrombopoietin (TPO), 5 ng/ml (±10%) IL-3 and 10 ng/ml (±10%) IL-6.After 6 days, cells are expanded in the same medium without TPO, IL-3and IL-6.

Alternatively, differentiation and expansion of erythroid cells can beperformed on a stromal cell line such as MS-5, using the same medium andcytokine combinations. This culture condition resulted in a generationof a higher percentage of more mature erythroid cells such asnormoblasts when compared to feeder-free conditions.

As described below, cell differentiation is preferably monitored bymorphological analysis of cytospins (i.e. cells were spun onto glassslides using a centrifuge so that cells can be stained and evaluatedmorphologically) and flow cytometry using anti-CD71 and anti-CD235aantibodies. Also as described below, one can analyze cell hemoglobincontent, preferably by flow cytometry using indirect staining withantibodies against human embryonic hemoglobin, fetal hemoglobin andadult hemoglobin and by PCR using embryonic, fetal and adulthemoglobin-specific primers.

After 5 days (±10%) of culture, the majority of live cells are erythroidprecursors and express CD71 and CD235a. After 10 days (±10%) of culture,most of the cells have a phenotype and a morphology of erythroid cells.By PCR, erythroid cells express high level of embryonic ζ- and fetalγ-hemoglobin, and low level of adult β-hemoglobin, as determined by PCR.After approximately 30 days of culture (±10%), essentially all of thelive cells (>95%) show positive stains with antibodies against fetal andadult hemoglobin, but no positive staining with antibodies againstembryonic ζ-hemoglobin. Following expansion, β-hemoglobin expressionincreased and ζ-hemoglobin expression decreased, and eventuallydisappeared by day 50 of culture, preferably as determined by PCR. Thecells are CD31− and CD34−. At this point, the cells are “erythroid cellsproducing adult-type β-hemoglobin” of the present invention.

With respect to CD31+ cells, Vodyanik et al. describes an earlyCD31+/CD34+/CD43+ hematopoietic progenitor and other CD34+ cellpopulations derived from hESC differentiation and embodies methods andCD34+ cell populations that are part of the present invention. VodyanikM, et al., “Leukosialin (CD43) defines hematopoietic progenitors inhuman embryonic stem cell differentiation cultures,” Blood 108:2095-2105(2006), incorporated herein by reference as if set forth in itsentirety.

EXAMPLES Example 1 Differentiation of hESCs into CD31+ Cells and/orCD34+

Differentiation of hESCs into CD31+ and/or CD34+ cells was achieved byco-culture of hESCs with a mouse OP9 bone marrow stromal cell line.

Undifferentiated hESCs (H1 and H9 lines; WiCell; Madison, Wis.) wereharvested by treatment with 1 mg/ml collagenase IV (Invitrogen) anddispersed by scraping to maintain the cells in small clumps. The hESCswere added to OP9 bone marrow stromal cells obtained from Dr. ToruNakano (Research Institute for Microbial Diseases, Osaka University,Japan, also available from ATCC) at a hESC density of 1.5×10⁶/20 ml per10 cm dish, or 0.3×10⁶/4 ml per well of a 6-well plate, in α-MEMsupplemented with 10% FBS (HyClone) and 100 μM MTG (Sigma).

The hESC/OP9 co-cultures were incubated for 6-7 days at 37° C. innormoxic conditions and 5% CO₂ with a half-medium change on day 4. Onday 6-7, cells were harvested and a single-cell suspension was preparedby treatment of the hESC/OP9 co-cultures with collagenase IV(Invitrogen; 1 mg/ml in α-MEM) for 20 minutes at 37° C. followed bytreatment with 0.05% trypsin-0.5 mM EDTA (Invitrogen) for 15 minutes at37° C. p The single-cell suspension was labeled CD31 paramagneticmonoclonal antibodies using Direct CD31 Microbead Kit (Miltenyi Biotech;Auburn, Calif.) as recommended by the manufacturer, and processedthrough an LS+ separation column attached to a Midi-MACS separation unit(Miltenyi Biotech) to obtain the magnet-retained fraction of purifiedcells. Alternatively, or in addition, the single-cell suspension waslabeled with CD34 paramagnetic monoclonal antibodies (Miltenyi Biotech).Purity of isolated CD31+ and/or CD34+ cells, as determined by flowcytometry, was generally greater than 95% at a single column run, andcell viability, as evaluated by Trypan blue exclusion, was always higherthan 95%.

Example 2 Large-Scale Expansion of Human Erythroid Progenitor Cells

To expand erythroid cells, CD31+ and/or CD34+ cells were cultured inSFEM (Stem Cell Technologies) supplemented with 0.3% of EX-CYTE(Serologicals Proteins, Inc.), 1 mg/ml iron saturated transferrin(Sigma), 10⁻⁶ M dexamethasone, and 20 ng/ml insulin in tissue cultureflasks coated with HEMA to prevent cell adherence. For the first 5 days,cells were cultured in the presence of 50 ng/ml SCF, 2 U/ml EPO, 50ng/ml TPO, 5 ng/ml IL-3 and 10 ng/ml IL-6. The subsequent incubationswere performed in the same media with insulin, transferrin,dexamethasone, SCF and EPO only, with medium changed every second day.

Cell differentiation was monitored throughout culture by morphologicalanalysis of cytospins and by flow cytometry using anti-CD71 (transferrinreceptor) and anti-CD235a (Glycophorin A) monoclonal antibodies.Hemoglobin analysis was performed by flow cytometry on cellspermeabilized with FIX&PERM® cell permeabilization reagent (Caltag;Burlingame, Calif.) using indirect staining with antibodies againsthuman embryonic hemoglobin ζ, fetal hemoglobin γ and adult hemoglobin βchains (Perkin Elmer; Norton, Ohio). In addition, hemoglobin expressionwas evaluated using PCR with primers specific for ζ-, γ- andβ-hemoglobin.

β-hemoglobin is adult hemoglobin. ζ-hemoglobin is embryonic and presentonly during embryonic development. γ-hemoglobin is usually presentduring the neonatal period and can be found in some conditions inadults. The presence of β-hemoglobin indicates that the erythroid cellsare not embryonic, as embryonic cells are β-hemoglobin negative.

After 5 days of culture, the majority of the cells were erythroidprecursors and expressed CD71 and CD235a (glycophorin). However, only afew cells were positive for hemoglobins. During next 5 days (day 10 ofculture), essentially all of the live cells in culture (>95%) hadphenotype and morphology of erythroid cells (CD71+/CD235a+) (FIGS. 1 and2). All cells contain embryonic (ζ) and adult (β) hemoglobins asdetermined by flow cytometry (FIG. 1) and by PCR.

After 50 days of culture, we observed at least a 2×10⁵ expansion oferythroid cells, and all erythroid cells showed positive stains withantibodies against fetal (γ) and adult (β) hemoglobin, but no positivestaining detected with antibodies against embryonic (ζ) hemoglobin (seeFIG. 1). By PCR, cells expressed adult β-hemoglobin and fetalγ-hemoglobin, but not embryonic ζ-hemoglobin. Morphologically, the cellpopulation consisted of nucleated erythroid cells at different states ofmaturation, including erythroblasts and normoblasts (FIG. 2). ThehESC-derived erythroid cells contained less than 0.1% of leukocytes asdetermined by staining with anti-CD45 monoclonal antibodies and were“essentially free of lymphocytes,” as determined by flow cytometry andRT-PCR for lymphoid markers.

Erythroid cells proliferated in culture for up to 60 days. After 60 daysthe cells stop proliferating and eventually died.

1. A method of culturing human embryonic stem cells into erythroid cellsproducing adult-type β-hemoglobin, the method comprising the steps of:(a) culturing human embryonic stem cells under conditions which favourdifferentiation of the cells into hematopoietic progenitors; (b)isolating human embryonic stem cell-derived cells selected from thegroup consisting of CD31+, CD34+, and CD31+/CD34+ cells, wherein thecells are enriched in CD31+/CD43+, CD31+/CD34+, CD34+/CD43+ orCD31+/CD34+/CD43+ hematopoietic progenitors; (c) culturing thehematopoietic progenitors to cause differentiation from CD34+/CD43+hematopoietic progenitors to erythroid precursors followed byself-renewal/expansion of the erythroid precursors and maturation of theerythroid precursors; and (d) recovering a population of maturederythroid cells, wherein essentially all of the live cells produce adultβ-hemoglobin and fetal γ-hemoglobin but not embryonic ζ-hemoglobin. 2.The method of claim 1, wherein the cells of step (d) are essentiallyfree of lymphocytes.
 3. The method of claim 1, wherein the cells of step(d) contain less than 0.1% leukocytes.
 4. The method of claim 1, whereinthe culturing of step (c) is in the presence of stem cell factor anderythropoietin under conditions preventing cell adherence.
 5. The methodof claim 1, wherein the human embryonic stem cells are co-cultured withstromal cells to produce the hematopoietic progenitors.
 6. The method ofclaim 1, wherein at least 0.2×10⁵ erythroid cells are generated from onehuman embryonic stem cell.
 7. The method of claim 1, wherein up to2.0×10⁵ erythroid cells are generated from one human embryonic stemcell.
 8. The method of claim 1, wherein the cells of step (d), whichproduce adult-type β-hemoglobin, are CD31−, CD34−, CD71+, CD235a+. 9.The method of claim 1, wherein the purity of the isolated hematopoieticprogenitors is greater than 95% at a single column run and cellviability, as evaluated by Trypan blue exclusion, is higher than 95%.10. The method of claim 1, wherein the CD31+/CD34+ cells comprise 30% to50% CD31+/CD34+/CD43+ hematopoietic progenitors and 50% to 70%CD31+/CD34+/CD43−/KDR^(bright) endothelial cells.
 11. The method ofclaim 1, wherein the cells express adult β-hemoglobin by 10 days (±10%)of culture, and wherein most of the cells have a phenotype and amorphology of erythroid cells.
 12. The method of claim 1, wherein afterapproximately 30 days (±10%) of culture, essentially all of the livecells (>95%) show positive stains with antibodies against fetalγ-hemoglobin and adult β-hemoglobin, but no positive staining withantibodies against embryonic ζ-hemoglobin.
 13. The method of claim 1,wherein after approximately 50 days (±10%) of culture, the cells of step(d) are CD31−/CD34− and show adult β-hemoglobin expression and noembryonic ζ-hemoglobin expression.
 14. The method of claim 1, whereinstep (c) is performed in serum-free culture conditions.